E. coli disease is an important and devastating disease to swine producers. Known for causing edema disease (ED) and post weaning diarrhea (PWD), the economic impact can be substantial with death losses as high as 50% (1). Multiple management and environmental factors have been associated with E. coli infections, including age at weaning, diet, crowding, and transportation. Certain host genetic factors, such as having receptors for the E. coli fimbriae to attach to the intestinal surface, also contribute to a pig's susceptibility to E. coli infection.
ED and PWD are both caused primarily by hemolytic E. coli proliferating in the small intestine. E. coli infection can also be diagnosed in pigs shortly after birth to two weeks of age (3). ED can occur in pigs between three and eight weeks of age and is characterized by subcutaneous and subserosal edema, a progressive ataxia, paralysis and a high mortality (2). PWD is commonly observed at 7-10 days post weaning but can occur up to eight weeks of age and is characterized by reduced growth rate, severe diarrhea, dehydration, toxemia, or death (2, 3).
Both PWD and ED can occur in the same group of pigs and the causative E. coli strains often share certain virulence factors. Usually the first manifestation seen with PWD is sudden death, as early as two days, after weaning. Pigs that do not die suddenly, display a decrease of feed consumption and watery diarrhea which leads to depression and life threatening dehydration. Many pigs show cyanotic discoloration of the tip of the nose, the ears, and the abdomen. Staggering and uncoordinated movements may also be seen in severely affected pigs. Peak mortality generally occurs 6-10 days after weaning. In a herd with PWD, morbidity can vary. Within a litter, the morbidity may be high and reach up to 80% with an average of 30-40%. Mortality in untreated herds can reach 26% (4).
Anorexia is often the first sign seen with edema disease. If diarrhea is to occur, it usually follows after the anorexia. The diarrhea usually disappears by the time the edema and nervous involvement become apparent. Edema can be seen in the eyelids, forehead, ears, and lips. Upon necropsy edema can be seen in the submucosa of the stomach, the mesocolon, gallbladder, and lungs. Progressive ataxia and mental confusion leading to complete recumbence and severe dyspnea are seen in the final stages. The mortality rate in edema disease can reach 50% to over 90% (4).
The source of E. coli in a weanling pig is usually derived from the environment either in the nursery, or the pig may acquire the E. coli in the farrowing unit and carry it into the nursery. Pathogenic E. coli can spread by means of aerosol, feed, farm vehicles, pigs, and other animals (4).
E. coli are part of the normal intestinal flora in pigs. Most of the intestinal E. coli do not possess the ability to cause disease. These E. coli pass through the intestines and are not able to attach to the intestinal wall and do not produce toxins. Those E. coli that are pathogenic and cause disease have the ability to do so because they have obtained genes which code for specific virulence factors (5). These virulence factors allow the E. coli to adhere to the intestinal wall, colonize the intestine, and produce enterotoxins, which can cause diarrhea and verotoxins which can cause edema disease.
Enterotoxigenic E. coli (ETEC) is the major type of E. coli implicated in diarrheal disease of pigs (FIG. 1) (5). These strains are characterized by their ability to adhere to the pig intestine and produce enterotoxins. Adherence to the intestinal tract is performed by frimbriae (pili) on the bacteria that attach to receptors located on the intestinal surface. These pili are highly antigenic filamentous protein structures that extend from the surface of the bacteria (5). The major pili found on ETEC are F4 (K88), F5 (K99), F6 (987p), F41, and F18.
Enterohemorrhagic E. coli (EHEC) is the major type of E. coli implicated in edema disease of pigs. The basis of colonization and toxin production is the same as with the ETEC. The only pilus that has been associated with edema disease in swine is F18, with the F18ab variant being associated more commonly with edema disease. The toxin produced by the EHEC is known as Stx2e. This toxin belongs to a family of toxins called shiga toxins or verotoxins. It is a high molecular weight protein that binds to specific receptors on vascular endothelial cells in certain target tissues (5). Therefore, the disease seen with EHEC is a result of toxemia. The receptor for the toxin is found in blood vessels in the brain, eyelid, stomach wall, mesentery of the colon, and the spinal cord (5). The toxin causes injury and death to the endothelial cells in these target organs.
Enteropathogenic E. coli (EPEC) strains, also known as attaching and effacing E. coli (AEEC) may play a role in diarrheal disease of pigs. These strains have only recently been investigated as a cause of diarrhea in weaned pigs and were first associated with diarrhea in humans (5). The EPEC cause disease by forming an attachment to the pig intestinal epithelial cells, possibly through the use of pili, and cause destruction of the microvilli (5). The lesions are called attaching and effacing lesions.
No universally effective prophylaxis is available for post weaning E. coli disease (4). Fundamental to the prevention of disease is to prevent villous atrophy and colonization (7). Villous atrophy and colonization are related to many factors such as rotavirus infection, diet, STb, and stress (7). Managerial factors that contribute to stress are changes in temperature, overcrowding, feed changes, humidity, and mixing of pigs.
No vaccines are currently commercially available for post weaning E. coli disease. However, there are companies that will prepare for each farm a killed or modified live vaccine using one attenuated strain of pathogenic E. coli found on the farm. This vaccine generally contains F18 or K88 pili but lacks the toxin genes. The attenuated strain is often grown on the farm and fed or given intranasal to pigs. Toxoids made from Stx2e are also used, but again are not commercially available. These vaccines are often not very pure and even though they may impact mortality due to E. coli disease, they generally do not decrease mortality to acceptable levels.
Egg immunoglobin, produced by hens that were vaccinated against fimbrial E. coli antigens, have also been developed as an antibody-containing egg powder in pig feed (4). The egg immunoglobin is produced by vaccinating the hen with an attenuated strain of ETEC or with fimbriae from the pathogenic E. coli strains. The egg yolks are then collected, and egg yolk antibody powder is then obtained by freeze drying the water soluble protein fraction of egg yolks (9). The theory was that it would provide immune protection against colonization with K88 and F18 positive E. coli (4). Marquardt et al. showed that egg-yolk antibodies were able to prevent experimentally induced ETEC diarrhea in 3 day old and 21 day old weaned pigs, and also decreased the occurrence of diarrhea in early-weaned pigs during a field trial study (9). Nevertheless, this is not always what is seen in the field, and producers seem to get mixed results using egg-yolk antibodies. What has been shown is that protection is only provided against challenge strains that have only the F18 fimbriae in common with the vaccine strains (4). Protection may also occur only for the strain of E. coli the hen was vaccinated for. Another drawback is that egg immunoglobin can be expensive to include in pig diets.
Some pigs are genetically resistant to ETEC and EHEC strains because they genetically lack the K88 and/or F18 pili receptor. Breeding for genetic resistance can help control E. coli disease. The difficult part about this process is the expense of testing and lack of tests available. A proprietary test for the presence of F18 receptors has been developed, but no test exists for the K88 receptor (6).
Antibiotics are often added to feed as a preventative. There are many drawbacks such as consumer acceptance and selection of resistant bacteria (4). Numerous antimicrobial substances are used for this purpose; some include: sulfonamide, trimethoprim, gentamicin, and other aminoglycosides. Isolates from ETEC and EHEC show the highest rate of resistance within swine E. coli, and this resistance is often induced within days or a few weeks (4).
Zinc oxide and spray dried porcine plasma included in weanling pig diets have also been used with mixed success.
Once an E. coli outbreak occurs, treatment must be administered to decrease mortality and morbidity. Antimicrobial therapy has been the treatment of choice. Antibiotics can be given parenterally or in the water once disease is detected. Antibiotic resistance with E. coli isolates is widely known. Pathogenic E. coli resistance has been detected against every antibiotic approved for use (4). Electrolytes can also be offered as a treatment choice but can be very costly to the producer.
One important factor that could result in the failure of current treatment and prevention techniques is the high genetic diversity of ETEC and EHEC strains. A high degree of heterogeneity has been shown among isolates from the same state and farm (8). Wilson, et al showed that serotypes associated with post-weaning diarrhea appear to be limited but have very diverse genetic backgrounds (10). This leads one to believe that multiple strains of the same virulence factors or serotype are the cause for a single outbreak of E. coli disease on a farm. The cause of heterogeneity is uncertain, but may be due to the fact that gene transfer can readily occur within swine E. coli. The fact that antibiotics, vaccines, and other treatments are always being used instigates the need of gene transfer for the survival of pathogenic swine E. coli. In addition, the great amount of fecal oral transmission in swine systems provides an environment needed for gene transfer to occur. This heterogeneity is evidence to explain why many traditional methods of treatment and prevention fail.
Therefore, what is needed is one or more isolated Bacillus microorganism that is capable of at least one of (A) inhibiting E. coli disease and (B) improving performance of an animal. A method of feeding swine one or more of the above-referenced Bacillus microorganisms to inhibit E. coli disease and/or improve performance of the swine is also needed. Additionally needed is a method of forming a direct-fed microbial from the above-referenced Bacillus microorganisms.